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The diagnosis and confirmation of human T cell lymphotropic virus (HTLV) type II infection has proven difficult, since most assays depend on antigenic cross-reactivity between HTLV-I antigens and HTLV-II antibodies. Type-specific HTLV infection rates were evaluated in a cohort of 233 injecting drug users screened with an HTLV EIA. Of the 52 EIA-reactive specimens, 48 were indeterminate or negative by standard Western blot. Type-specific HTLV results determined by polymerase chain reaction (PCR) were 0, HTLV-I; 92%, HTLV-II; 6%, type indeterminate; and 2%, negative. Among 42 EIA-reactive, HTLV-II-PCR-positive individuals tested by a p21 envr Western blot, all were positive and 74% had antibodies to the tax protein. This study found a high rate (22.3%) of HTLV reactivity, with HTLV-II usually the sole responsible agent; shortcomings in standard HTLV-I-based diagnostics but usefulness of PCR and p21 envr Western blots for typing and confirmation of HTLV reactivity; and a high prevalence (74%) of anti-tax antibody among HTLV-II-seropositive subjects, suggesting increased potential for infectivity.